The B cell response to antigen is regulated by a variety of co-receptors that convey information to the B cell about the quality of the antigen and the status of the ongoing immune response. Over the last year we focused our attention of two potent regulators of B cell responses, namely the CD19/CD21 complex and the Fcgamma family of receptors. Signaling through the B cell receptor (BCR) is both amplified and prolonged by coligation of the BCR and the CD19/CD21 complex through the binding of complement fixed antigens. The low affinity Fc receptor, FcgammaRIIB, is a potent B cell inhibitory receptor and as such plays a central role in controlling antibody-mediated autoimmunity. Determining how these co-receptors influence BCR-induced signaling should add fundamentally to our understanding of the mechanism by which B cells are activated. Over the last year we have made progress in defining the mechanisms by which the positive coreceptor, the CD19/CD21 complex and the inhibitory receptor, FcgammaRIIB, function to regulate B cell signaling. In addition we have initiated studies to determine the function of the Fc like receptor 4 (FcRL4) present on a subset of atypical MBCs observed to be greatly expanded in HIV infected individuals with high virus levels and in children and adults in malaria-endemic areas. Our earlier studies provided biochemical evidence that the B cell coreceptor, the CD19/CD21 complex, when coligated to the BCR through the binding of complement tagged antigens prolongs and enhances BCR signaling in part by prolonging the association of the BCR with sphingolipid- and cholesterol-rich membrane microdomains, termed lipid rafts. We also provided biochemical and genetic evidence that the CD81 component of the CD19/CD21 complex was essential for the raft stabilizing function of the CD19/CD21 complex. We showed that CD81 associates with raft lipids upon coligation of the BCR and the CD19/CD21 complex and that in B cells from CD81-deficient mice coligated BCR and CD19/CD21 complexes failed to associate with raft lipids or enhance BCR signaling. We further demonstrated that upon coligation CD81 was palmitoylated and that the palmitoylation was essential for its raft-stabilizing function. Thus, we defined a novel mechanism by which a co-receptor influences the local lipid environment of a receptor namely by inducible lipidation. Over the last year using mutant CD81 in which the six cysteines that are modified by palmitoylation were changed to alanine, we demonstrated that palmitoylation was not necessary for interactions of CD81 with themselves but was essential for the interaction of CD81 with a raft lipid probe and for complete downstream signaling from the BCR when coligated to CD19/CD21. In these studies lipid rafts were operationally defined by their relative detergent insolubility, due to the tight packing of the saturated chains of the raft lipids and by their dependence on cholesterol. However, the use of detergents and cholesterol-depleting drugs are fraught with potential artifacts including creating the lipid heterogeneities we set out to study. High resolution fluorescence resonance energy transfer (FRET) coupled with total internal reflection microscopy (TIRFM) or confocal microscopy offered the opportunity to quantify the interactions of the BCR with raft lipids in live cells over the time and length scale necessary to capture the earliest events in antigen-initiated B cell activation. To study the interactions of raft lipids with BCRs we generated cell lines that expressed a BCR containing the FRET donor fluorescent protein CFP and the FRET acceptor protein, YFP, tethered to the membrane by either raft lipids or by non-raft lipids. FRET confocal imaging of living B cells revealed that within seconds of antigen binding the BCR selectively and transiently associated with the lipid raft constructs and that this association was prolonged by coengagement of the BCR and the CD19/CD21 coreceptor complex. Using live cell FRET TIRF imaging we recently provided direct evidence that the association of the BCR with raft lipids following antigen binding is blocked by the FcgammaRIIB. Over the last year we extended these studies to investigate the interaction of the FcgammaRIIB and BCR and raft lipids when coligated by the binding of immune complexes (ICs). We discovered that the binding of ICs induced the colocalization of the BCR and FcgammaRIIB in microscopic clusters but that within these clusters the BCR and Fcgamma RIIBs were not in close molecular proximity as measured by FRET. Nonetheless, binding of ICs resulted in a block in the earliest events in BCR signaling beginning with the oligomerization of the BCR into immobile signaling active clusters. Following IC binding the BCRs failed to associate with a lipid raft probe in contrast to the FcgammaRIIB that stably associated with the raft lipid probe. The ability of the FcgammaRIIB to block the initiation of BCR signaling appeared to be dependent on its ability to associate with raft lipids as mutant FcgammaRIIBs that are unable to associate with lipid rafts did not block BCR signaling. Taken together these studies indicate that the FcgammaRIIB affects BCR signaling at a much earlier point than previously appreciated. In summary, FRET measurements in TIRF microscopy are providing the first direct evidence for the antigen-induced association of the BCR with lipid rafts in living cells and the regulation of this very early event by the CD19/CD21 complex and the FcgammaRIIB. Over the last year we initiated studies to define the function of FcRL4s receptor originally described to be expressed on a subpopulation of memory B cells termed tissue-like MBCs. FcRL4 was also recently discovered on a population of MBCs termed atypical or exhausted MBCs found in HIV infected individuals and in individuals living in malaria endemic areas. The FcRL4 has no known ligand and contains in its cytoplasmic domain an immunoregulator tyrosine inhibitory motif (ITIM) that has been shown to be functional when expressed as part of a chimeric FcgammaRIIB receptor. We learned that when transfected into human B cell lines the FcRL4 functions to block BCR signaling at the point of Syk recruitment. Thus, FcRL4 functions in a ligand independent fashion to limit BCR signaling. This finding has important implications for the mechanism by which B cells may become refractory to signaling but remain viable.